Malaria vector mosquitoes possess an innate immune system that can recognize and respond to infection by malaria parasites. Probably the most active immune compartment of the mosquito is the hemolymph, a serum of soluble proteins that fills the open body cavity. Malaria ookinetes, oocysts, and sporozoites must survive as yet uncharacterized immune factors in the hemolymph that can destroy them by melanotic encapsulation or other mechanisms. Sporozoites must migrate within hemolymph and invade the salivary glands in order for transmission to occur. The parent grant to this proposal focuses on the biology of malaria sporozoites in the mosquito, particularly the identification of receptors for salivary gland invasion. Work under the parent grant has shown that exposure to the hemolymph compartment causes rapid destruction of the majority of sporozoites by an unknown mechanism, which fewer than 20% escape to invade the salivary glands. Due to technological limitations, however, the critical hemolymph compartment has been a little-studied period of the malaria life cycle. Hemolymph is particularly well suited for proteomic characterization because the functional immune response in this key compartment is based on the interplay between many soluble proteins (1,500-3,000 total) that are synthesized elsewhere by a variety of cell types, and thus cannot be described accurately either spatially or temporally by mRNA-based assays. The proposed project will leverage new technological advances in proteomics and the new genome sequences of parasite and vector to generate the first (to our knowledge) proteomic datasets from an infectious disease vector. To this end, we propose two Aims: 1) Identify the functional proteome of mosquito hemolymph in the resting state using new approaches in liquid chromatography-tandem mass-spectrometry for sequencing of purified Anopheles gambiae hemolymph. 2) Carry out differential expression analysis to identify hemolymph proteins that are induced by malaria sporozoite infection. The generated datasets will be published and made publicly available by web archive. Understanding the mosquito anti-parasite immune response could offer new vector-based malaria control opportunities. Moreover, the innate immune response of invertebrates is clearly the phylogenetic precursor of mammalian innate immunity, with structural and functional conservation described for a growing number of molecules. It is possible that recognition and signaling mechanisms involved in innate immune activation by sporozoites in mosquitoes may be similarly conserved in mammals, which could be helpful in vaccine development.